Currently used methods of microbial identification have numerous shortcomings. Such shortcomings include the need for special growth media, the inability to distinguish closely related species and strains, the need for large amounts of sample, the need to culture the organism for a lengthy period of time, and the like.
The sequence of 16S ribosomal RNA has long been use for analyzing the evolutionary relationship between microorganisms. Many groups have used differentially hybridizing DNA probes (or batteries at such differential probes) in order to identify unknown microorganisms on the basis of hybridization to ribosomal RNA. However, nucleic acid hybridization is an imprecise technique and is ill-suited for distinguishing between closely related strains or species.
The DNA sequences of 16S RNA genes in public databases such as GENBANK have serious problems with regards to accuracy and completeness. These problems arise because many bacteria have two or more 16S ribosomal RNA genes and sequence variations occur between the different copies of the gene present in the same genome, i.e., polymorphisms. Traditional analysis of 16S ribosomal RNA is involves the cloning and sequencing of individual 16S ribosomal RNA genes. In many instances, no publicly available information exists as to the sequences of all of the 16S ribosomal RNA genes in given organism. A similar lack of information exists with respect to other ribosomal RNA genes in bacteria and other microorganisms.
In view of the shortcoming of the commonly used microbial identification systems, it is of interest to provide new gene sequence by sex methods for the identification and classification of unknown microorganisms.